Self-assembling synthetic proteins

ABSTRACT

The present disclosure provides for a synthetic immunogenic protein for use as an immuno-modulatory agent to enhance mammalian immune reactions towards conjugated protein or peptide containing antigens that are otherwise poorly immunogenic, including but not limited to self-antigens. The chimeric immunogenic proteins of the present disclosure can be used in the treatment of many illnesses, including but not limited to cancers, infectious disease, autoimmune disease, allergies and any clinical indication involving or affected by the immune response of a mammalian host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication No. PCT/IB2014/001125, entitled SELF-ASSEMBLING SYNTHETICPROTEINS, filed on Mar. 17, 2014, which in turn claims priority to U.S.Provisional Application No. 61/791,268 filed Mar. 15, 2013, which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to generation of a synthetic proteinscaffold which exhibits specific functional and biophysicalcharacteristics advantageous in the use of the synthetic protein todeliver and present antigens to the host immune system, even when saidantigens are poorly or non-immunogenic in the host, such that the hostis induced to raise a specific antibody response towards the antigens. Asynthetic protein that is immunogenic to mammalian immune systems, andwhich can assemble into stable defined multimers is described. Further,a method for the use of said protein to confer immunogenicity and inducespecific antibody responses to poorly or non-immunogenic peptides isdescribed.

BACKGROUND OF THE INVENTION

The introduction of a foreign (non-self) substance, i.e. an antigen, tothe immune system of a vertebrate usually results in the induction of animmune response by the host against that antigen. Typically this willinvolve the stimulation of B and/or T-lymphocytes, and the production ofimmunoglobulin molecules (antibodies) that recognise and bind to theantigen. There are a great many factors that influence the extent towhich a substance will induce an immune response in a host. The degreeof foreignness is important as the immune system has evolved anddeveloped to be non-responsive to ‘self’. Size is also a significantfactor, with larger molecules generally being more immunogenic thansmaller ones. Molecules below ˜1000 Da molecular weight (classified ashaptens) are too small to be seen by the immune system in isolation andare therefore non-immunogenic, though they may still be antigenic.

Larger molecules will be more complex and therefore more likely tocontain multiple immunogenic epitopes, and are also more readilyengulfed and processed by antigen presenting cells (APCs). Thecomposition of the substance is also important, with proteins easilybeing the most immunogenic. Polysaccharides are much less immunogenic(in isolation) and nucleic acids and lipids are essentiallynon-immunogenic. Similarly, particulate or denatured antigens are moreimmunogenic than soluble and native molecules. The route of exposure andbiological activities of foreign substances can also significantlyaffect the nature and extend of any immune response by the host. Forexample, parenteral injection of a substance that interacts withcomponents or cells of the immune system will result in a much strongerresponse than mucosal exposure (ingestion/inhalation) of a relativelyinert or inactive substance.

T-cells and B-cells recognise and respond to foreign antigens indifferent ways. Specialised antigen presenting cells or APC's(macrophages, dendritic cells and B-cells) continually interrogate theirenvironment by taking up molecules from the extracellular space,including macro-molecules and whole micro-organisms, and processing theprotein content of these. Exogenous proteins are digested by a panel ofprotease enzymes in endosomes, and the resulting peptides displayed onthe surface of the cells in the groove of MHC II molecules. These inturn are recognised by specialist receptors on the surface of T-cells(TCRs). The process of T-cell development ensures that those T-cellsdisplaying receptors that react to MHC II containing self-peptides aredepleted, and only those that recognise foreign sequences maturesuccessfully. The peptides recognised by T-cells (T-cell epitopes) areinvariably linear, but are not always exposed or accessible on thenative folded protein from which they were derived.

In contrast, the B-cell surface receptors or immunoglobulins (BCR)recognise and interact primarily with soluble proteins (bothconformational and denatured epitopes), haptens, polysaccharides, and toa lesser extent some lipids and nucleic acids. The specificity of a BCRis identical to that of the antibody that the B-cell can secrete. Uponbinding of its cognate antigen, the BCR is internalised and the boundantigen processed. Only when it is a protein, or is attached to aprotein component, will it then be presented on the cell surface as partof an MHC II complex. Under these conditions, the B-cell is thenavailable to be stimulated by a T-helper cell that has a TCR recognisingthe presented peptide. In the case of a large or complex protein, aB-cell can therefore be activated by a number of different T-cells, noneof which need necessarily recognise the same antigenic epitope as theBCR, but all of which will recognise a peptide component of the sameprotein. It is this capacity of vertebrate immune systems that allowsthem to develop antibodies against antigenic determinants that are notin themselves immunogenic.

In order to develop effective vaccines, it is necessary to presentantigenic epitopes to the host immune system in such a way as tostimulate a strong immune response, involving both T- and B-lymphocytes.Immune responses that do not involve activation of effector (helper)T-cells and subsequent stimulation of B-cells by these are usuallyshort-lived and do not result in antigenic memory, i.e. do not lead to amore aggressive and more rapid antibody response when the host isexposed to the immunogen for a second time.

It is also often a requirement of vaccines to illicit antibodies thatare able to inhibit, block or otherwise neutralize the functionalactivity of the target, and so afford protection to the host. This canpresent a major challenge for many reasons. Frequently, those epitopesthat need to be targeted by an antibody response have not beenidentified due to a lack of structure-function data relating to thetarget. Even when detailed information relating to the target and itsinteractions, the identified epitope(s) may not be immuno-dominant andtherefore might not generate the responses sought in the majority ofpatients. In other cases, the key protective antigenic determinantsmight not be protein, e.g. polysaccharides on pathogen glyco-proteins,and so not immunogenic (T-cell dependant) in isolation.

The vast majority of vaccines are delivered through parenteral routes;however there are many advantages to mucosal delivery such as patientcompliance, self-administration, reduced risk of infection, and thepossibility of inducing both mucosal and systemic immunity. There arealso many obstacles to overcome, such as vaccine dilution, the presenceof micro-flora, the need to withstand low pH when given orally, to crossmembranes and the need for potent adjuvants (Vajdy et. al., 2004).Moreover, mucosal administration can lead to B-cell tolerance ratherthan an immune response. The dosage can also have a major effect onimmune responses. If an immunogen is not effectively cleared by theimmune system, or if the system is swamped by too high a dose, thentolerance can be induced. Conversely, too low a dose can also lead totolerance, or simply fail to stimulate sufficient immune cells.

A number of approaches have been developed to help overcome thesedifficulties. In most cases, vaccines are administered along with someform of adjuvant. Adjuvants are essentially any formulation that, whenadministered together with an immunogen, causes one or more ofpersistence of the immunogen at the site of injection, enhancement ofco-stimulatory signals, non-specific stimulation of lymphocyteproliferation or granuloma formation. They come in a variety of forms,for example Freund's complete adjuvant consists of inactivatedMycobacterium whilst others comprise an emulsion of oil (e.g. squalene)in water. These are most commonly used in animals as they can causeadverse reactions at the injection site. Some organic adjuvants are usedin human vaccines such as Montanide© (mineral oil based with vegetablecomponents) though more commonly they are inorganic such as aluminiumsalts.

Amongst the most popular and widely adopted methods to overcome lowimmunogenicity has been to couple an identified or desired antigen orantigenic determinant with a strongly immunogenic carrier. This is aprotein or polypeptide derived from a different species, for exampleBovine Serum Albumin (BSA) and Keyhole Limpet Hemocyanin (KLH) are oftenused as carriers of chemically conjugated haptens and small peptides togenerate antibodies in animals (Berzofsky and Berzofsky 1993). Thecarrier presents the haptens on a molecule large enough to be seen andprocessed by the host immune system, and also stimulates the host immuneresponse by being inherently immunogenic.

Generally speaking, carrier proteins derived from sources morephylogenically distant to the recipient are better. The carrier is thenlikely to be more different from host proteins and hence more foreign. Afurther important consideration when selecting a carrier protein is thepossibility that if it is a homologue of a host protein, and so sharessignificant homology, then the elicited immune response might also reactwith host proteins and lead to adverse side effects. Non-proteinantigens can only be coupled chemically, which may limit control overwhere on the carrier they are attached and how they are presented. Smallpeptides can be coupled chemically or genetically. In other areas ofresearch, modern developments in bioinformatics have led to an increasein the rational design of immunogens, and in particular of peptides.

The concept of peptide vaccines is based on identification and chemicalsynthesis of B-cell and T-cell epitopes which are immunodominant and caninduce specific immune responses, for example coupling a B-cell epitopeof a target molecule to a widely recognised T-cell epitope to make itimmunogenic (Naz R. K. and Dabir P. 2007). Peptides are seen as beingrelatively easy to produce when compared to larger and more complexprotein antigens. They can also possess favourable chemical stability,and lack oncogenic or infectious potential making them attractivevaccine candidates. However, several obstacles limit the widespreadusefulness of peptide vaccines including their often low inherentimmunogenicity and the need for better adjuvants and carriers. Otherresearch has suggested that recombinant chimeric proteins may be mademore immunogenic if T helper epitopes are incorporated as tandem repeats(Kjerrulf M, et al. 1997).

Another popular class of carrier protein is bacterial toxoids. In thecase of vaccines against bacterial infection where the symptoms ofinfection are caused by the action of toxins, then these can be used asthe vaccine itself. It is of course necessary to render them inert,either chemically or by the use of a non-toxic component. Suchattenuated toxins e.g. diphtheria and tetanus vaccines that weredeveloped in the 20^(th) century are called toxoids.Polysaccharide-protein conjugate vaccines in use or late stagedevelopment by companies such as Wyeth (Pfizer), Aventis Pasteur, GSK,Merck and others use tetanus, diphtheria or other toxoids.

The B sub unit of Cholera toxin or E. coli heat labile enterotoxin (LT)have been proposed by many as a useful carrier proteins for variousvaccine applications (Nemchinov, L. G et al. 2000, George-Chandy, A. etal. 2001, U.S. Pat. No. 6,153,203). It is highly immunogenic, and in theabsence of the CT-A sub unit is non-toxic. Forming the basis of a widelyused Cholera vaccine it has a demonstrated safety profile when usedsystemically. Whilst relatively small (˜12 kDa), it can assemble intostable pentamers giving it a much higher molecular weight.

Of particular interest to many researchers is to exploit the affinity ofCTB and enterotoxin pentamers to G_(M1) ganglioside, a branchedpenta-saccharide found on the surface of nucleated cells. During cholerainfection, it is this binding that facilitates the translocation of theholotoxin across the intestinal epithelium. There have been numerousreports in the literature that vaccines based on CTB fusions, chemicalor genetic, can be effective at stimulating mucosal immunity(George-Chandy, A. et al. 2001, Houhui Song et al. 2003, Shenghua Li etal. 2009, Harakuni, A. et al. 2005) when administered orally orintra-nasally. In order to retain the ability to react with G_(M1)ganglioside, which binds at the pocket formed between adjacent CTBsubunits, it is essential that the target antigen does not block accessto the G_(M1) binding site and does not prevent the assembly of CTBmultimers.

It has been demonstrated that genetic fusions can be made to CTB thatsuccessfully retain G_(M1) binding, however there are also limitations.Liljeqvist, S. et al. (1997) showed that the serum albumin-bindingdomain of Strepococcal protein G could be fused genetically to either N-or C-terminus of CTB, or to both termini simultaneously, and retainG_(M1) binding. It was noted however that the N-terminal fusion and thedual fusion proteins were significantly less efficient at forming stablepentamers, and less effective in binding to G_(M1). Similarly, it hasbeen demonstrated that large genetic fusions are unable to formpentamers unless a heterogenic mixture of both chimeric and wild typeCTB are present (Harakuni, A. et al. 2005).

SUMMARY OF THE INVENTION

According to the disclosure, the highly immunogenic nature of asynthetic carrier is able to increase the host immune response to theincluded variable sequences due to its inherently immuno-stimulatory andadjuvant-like properties. It is further disclosed that the syntheticcarrier according to the disclosure will cause the host to raise anantibody response to ‘self’ antigenic determinants encoded at least inpart by the variable sequences.

In an illustrative embodiment, the recombinant synthetic protein is ableto assemble into stable homo-pentamers, wherein each monomer includesone or more antigenic determinants derived from target proteins.

In another illustrative embodiment, the recombinant synthetic protein isable to assemble into stable hetero-pentamers, wherein monomersexpressing different antigenic determinants are assembled together.

In a further illustrative embodiment, the recombinant synthetic proteinis substantially similar to the following sequence:

(SEQUENCE ID: 1) FTDIITDICGEYHNTQIHTLNDKILSYTESLVGKREIILVNFKGGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLSNSKIEKLCVWNNKTPHSIAAIS MVR

In yet a further illustrative embodiment, the recombinant syntheticprotein includes linker or spacer sequences, whereby the variablesequences positioned at one or both termini of the synthetic carrier areseparated from the synthetic carrier in such a way as to enable thesynthetic carrier elements from several recombinant proteins toassociate. In one illustrative embodiment the recombinant syntheticprotein having a linker sequence that attaches to a growth factor issubstantially similar to the following sequence:

(SEQUENCE ID: 2) ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCSGGSGGTSGGGGGSGFTDIITDICGEYHNTQIHTLNDKILSYTESLVGKREIILVNFKGGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLSNSKIEKLCVWNNKTPHSIAAISMVR 

In another illustrative embodiment the recombinant synthetic proteinwill associate into stable pentamers in the form of a ring. Othermultimeric assemblies, for example but not limited to dimers, trimers,tetramers and larger multimers are also envisaged within the scope ofthe disclosure. The linker sequences can be varied, and as a minimumshould be of sufficient length to prevent the variable antigenicdeterminant sequences from sterically inhibiting assembly of multimersby association of synthetic carrier domains. It is contemplated withinthe scope of the disclosure that the linkers or spacers can be flexibleallowing for the formative of stable pentamers.

In another embodiment, the pentameric structure of the multimer isstabilized by the introduction of at least one stabilizing molecule. Thestabilizing molecule can be co-expressed in the same cells as thesynthetic molecule, or alternatively can be added exogenously.

In one embodiment, the stabilizing molecule includes sequencessubstantially similar to the CT-A2 domain of cholera toxin.

In another embodiment, the stabilizing molecule comprises sequences thatinteract with the synthetic protein such as to stabilise the multimers.It is further contemplated that this stabilization may result in agreater proportion of pentameric forms using recognized manufacturingtechniques known in the art.

In a further illustrative embodiment, the stabilizing molecule can serveto anchor an antigenic domain to the multimeric form of the syntheticprotein.

In yet a further embodiment, interrupted repeats of the stabilizingmolecule can serve to associate two or more multimeric forms of thesynthetic protein.

In an illustrative embodiment, the linkers or spacers sequences will begenerally hydrophilic, and be flexible such that they include no definedsecondary structure.

In another illustrative embodiment, the linker sequences will such thatthey do not directly or indirectly influence the folding of thesynthetic carrier domain, or of the variable antigenic determinantdomains.

In a further illustrative embodiment, the linker sequences will beresistant to proteolysis by extracellular proteases.

In another illustrative embodiment, the linker or spacer sequencesinclude but are not limited to the following: SSG, SSGGG, SGG, GGSGG,GGGGS, SSGGGSGGSSG, GGSGGTSGGGSG, SGGTSGGGGSGG, GGSGGTSGGGGSGG,SSGGGSGGSSG, SSGGGGSGGGSSG, SSGGGSGGSSGGG, and SSGGGGSGGGSSGGG

In one illustrative embodiment, the linker sequences will includesequences that, either alone or in conjunction with flanking sequencesfrom the variable antigenic determinants or synthetic carrier, will formT-cell epitopes. Preferably, the T-cell epitopes will be T-cell epitopesas recognised by human T-helper cells when bound by MHC (MajorHistocompatibility Complex) class II proteins. Preferably, the T-cellepitopes will be bound by MHC II molecules of the HLA (human leukocyteantigen)-DR sub-class.

In another illustrative embodiment the synthetic recombinant protein isstructurally homologous to B-sub unit of the A1B5 group of bacterialholotoxins.

In a further illustrative embodiment, multimers of the recombinantsynthetic protein are able to bind to G_(M1) ganglioside.

In an illustrative embodiment, the synthetic recombinant protein is animmunogenic protein that induces an immune response in mammalian hosts.

In another illustrative embodiment, the synthetic recombinant protein isan immunogenic protein including one or more variable sequences thatrepresent antigenic determinants to which it is desirable to generate animmune response by patients. The variable sequences can be located atthe N- and/or C-terminus of the sequence encoding the synthetic carrier,or can be incorporated within the synthetic carrier encoding sequencesuch that they are presented appropriately to the cells of the immunesystem. In a preferred embodiment the variable sequences are presentedin such a way as to replicate the conformation that the sequencesdisplay in the natural molecules from which they are derived, and areaccessible to the immunoglobulin cell-surface receptors of B-cells.

In another illustrative embodiment, the variable antigenic determinantsare derived from signalling molecules, including but not limited togrowth factors such as adrenomedullin (AM), angiopoietin (Ang), bonemorphogenetic proteins, epidermal growth factor (EGF), fibroblast growthfactor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor(IGF), nerve growth factor (NGF) and other neurotrophins,platelet-derived growth factor (PDGF), transforming growth factoralpha(TGF-α), transforming growth factor beta(TGF-β),tumor_necrosis_factor-alpha(TNF-α) and vascular endothelial growthfactor (VEGF).

In another embodiment, the variable antigenic determinants are derivedfrom other ligand molecules involved in the development or progressionof disease, such as, but not limited to PDL1.

In another embodiment, the variable antigenic determinants are derivedfrom receptors found on the surface of cells, and involved in signalingevents that regulate the growth of cells such as the natural receptorsof growth factors.

In a further embodiment, the variable antigenic determinants are derivedfrom tumor antigens.

In yet another embodiment, the variable antigenic determinants arederived from bacterial, viral, fungal or other pathogens.

BRIEF DESCRIPTION OF DRAWINGS

The embodiments described in the present disclosure are illustrated inthe figures of the accompanying drawings which are meant to be exemplaryand not limiting, in which like references are intended to refer to likeor corresponding parts, and in which:

FIG. 1. Illustrates the ELISA signals produced by a panel of solublemutant clones binding to an immobilized galactose-containing molecule(GM1), following 3 rounds of selection;

FIG. 2. Illustrates a Western blot of soluble mutant proteins from oneof the mutant libraries electrophoresed on an SDS gel, some of whichretain the ability to form pentamers, and others that do not;

FIGS. 3A and 3B. Illustrate a selection of mutations identified afterselecting and screening several mutant libraries FIG. 3(a) Sequences ofa selection of mutants, each isolated from one of 7 separate mutantlibraries and illustrating the distribution of mutated residues, andFIG. 3(b) sequences of synthetic proteins derived from compilingmutations derived from several different mutant clones;

FIG. 4. Illustrates GM1-binding ELISA data from four compilationmutants, showing that two clones have retained galactose bindingactivity, and two have not;

FIG. 5. Illustrates the structure of the complete cholera holotoxinmolecule;

FIG. 6. Illustrates sequences of the synthetic protein according to thedisclosure (Sequence ID: 1); and a further sequence of synthetic proteinaccording to the disclosure comprising sequences encoding human TGFBeta1 (Bold), a flexible linker (Italicised), and synthetic proteincarrier (Sequence ID: 2);

FIG. 7. Illustrates the distribution of targeted residues in severalmutant libraries (a), and sequences of synthetic proteins (templates)successively obtained (b);

FIGS. 8A and 8 Illustrate GM1-binding ELISA (a) and Western blot (b) ofsynthetic carrier (SEQUENCE-3: 5B5−/+G33D) and its immediate precursor(1B6). Specific detection was via cMyc-tag;

FIGS. 9A and 9B. Illustrate impaired detection of synthetic carrier byanti-CTB monoclonal antibody (a) and polyclonal anti-CTB serum (b);

FIG. 10. Illustrates immunogenicity of synthetic carrier (precursor)fused to hFGF2. Depicted are IgG responses to hFGf2, in mice sera 28days after immunisation;

FIGS. 11A and 11B. Illustrate GM1-binding ELISA (a) and culture mediumand crude periplasm preps signals on Western blot (b) of carrier5B5_I74D, potentially deficient in assembling with CTA2. Including 5B5,5B5_G33D and 5B5_T78D;

FIG. 12. Illustrates sequences of the synthetic protein according to thedisclosure comprising amino acid sequence (Sequence ID: 3) of syntheticcarrier protein indicated are glycine-33 and isoleucine-74 (bold andunderlined), resulting in absence of GM1-binding and assembly with CTA,respectively, when changed to an aspartate (G33D and I74D insequence-4); and

FIG. 13. Illustrates a further sequence of synthetic protein accordingto the disclosure (Sequence ID:4) comprising amino acid sequence ofsynthetic protein comprising sequences encoding synthetic carrier, aflexible linker (in italics), and human growth factor FGF2 (underlined).

DETAILED DESCRIPTION

Detailed embodiments of the present recombinant proteins or vaccines aredisclosed herein, however, it is to be understood that the disclosedembodiments are merely exemplary, which may be embodied in variousforms. Therefore, specific functional details disclosed herein are notto be interpreted as limiting, but merely as a basis for the claims andas a representative basis for teaching one skilled in the art tovariously employ the recombinant protein disclosed herein.

The present disclosure provides a synthetic recombinant protein forimproving the presentation of the maximum number of growth factorepitopes, tumor antigen epitopes, and/or receptor binding sites aselements of an immunogenic recombinant protein.

In one illustrative embodiment, a synthetic recombinant protein as shownin Sequence ID: 2 containing at least one growth factor including butnot limited to human transforming growth factor (TGF), a tumor antigen,and/or a receptor is described. In alternative illustrative embodiments,the protein may express other immunogenic recombinant proteins that aremodeled based upon known immunogenic proteins. It is contemplated withinthe scope of the disclosure that such recombinant proteins will beexpressions of polypeptides that are highly immunogenic to the humanimmune system. Preferably, the recombinant proteins confer additionalproperties to the chimeric protein, for example, high expression yieldand ease of manufacture, oral stability and the ability to cross fromgut to blood stream, and/or previous safe use in humans.

Certain illustrative embodiments as provided herein include recombinantproteins according to the disclosure within vaccine compositions andimmunological adjuvant compositions, including pharmaceuticalcompositions, that contain, in addition to recombinant proteins at leastone adjuvant, which refers to a component of such compositions that hasadjuvant activity.

An adjuvant having such adjuvant activity includes a composition that,when administered to a subject such as a human (e.g., a human patient),a non-human primate, a mammal or another higher eukaryotic organismhaving a recognized immune system, is capable of altering (i.e.,increasing or decreasing in a statistically significant manner, and incertain preferred embodiments, enhancing or increasing) the potencyand/or longevity of an immune response. In certain illustrativeembodiments disclosed herein a desired antigen and or antigens containwithin a protein carrier, and optionally one or more adjuvants, may soalter, e.g., elicit or enhance, an immune response that is directedagainst the desired antigen and or antigens which may be administered atthe same time or may be separated in time and/or space (e.g., at adifferent anatomic site) in its administration, but certain illustrativeembodiments are not intended to be so limited and thus also contemplateadministration of recombinant protein in a composition that does notinclude a specified antigen but which may also include but is notlimited to one or more co-adjuvant, an imidazoquinline immune responsemodifier.

Accordingly and as noted above, adjuvants include compositions that haveadjuvant effects, such as saponins and saponin mimetics, including QS21and QS21 mimetics (see, e.g., U.S. Pat. No. 5,057,540; EP 0 362 279 B1;WO95/17210), alum, plant alkaloids such as tomatine, detergents such as(but not limited to) saponin, polysorbate 80, Span 85 and stearyltyrosine, one or more cytokines (e.g., GM-CSF, IL-2, IL-7, IL-12,TNF-alpha, IFN-gamma), an imidazoquinoline immune response modifier, anda double stem loop immune modifier (dSLIM, e.g., Weeratna et al., 2005Vaccine 23:5263).

Detergents including saponins are taught in, e.g., U.S. Pat. No.6,544,518; Lacaille-Dubois, M and Wagner H. (1996 Phytomedicine2:363-386), U.S. Pat. No. 5,057,540, Kensil, Crit. Rev Ther Drug CarrierSyst, 1996, 12 (1-2):1-55, and EP 0 362 279 B1. Particulate structures,termed Immune Stimulating Complexes (ISCOMS), comprising fractions ofQuil A (saponin) are haemolytic and have been used in the manufacture ofvaccines (Morein, B., EP 0 109 942 B1). These structures have beenreported to have adjuvant activity (EP 0 109 942 B1; WO 96/11711). Thehaemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A)have been described as potent systemic adjuvants, and the method oftheir production is disclosed in U.S. Pat. No. 5,057,540 and EP 0 362279 B1. Also described in these references is the use of QS7 (anon-haemolytic fraction of Quil-A) which acts as a potent adjuvant forsystemic vaccines. Use of QS21 is further described in Kensil et al.(1991. J. Immunology 146:431-437). Combinations of Q S21 and polysorbateor cyclodextrin are also known (WO 99/10008). Particulate adjuvantsystems comprising fractions of QuilA, such as QS21 and QS7 aredescribed in WO 96/33739 and WO 96/11711. Other saponins which have beenused in systemic vaccination studies include those derived from otherplant species such as Gypsophila and Saponaria (Bomford et al., Vaccine,10 (9):572-577, 1992). [0203] Escin is another detergent related to thesaponins for use in the adjuvant compositions of the embodiments hereindisclosed. Escin is described in the Merck Index (12.sup.th Ed.: entry3737) as a mixture of saponin occurring in the seed of the horsechestnut tree, Aesculus hippocastanum. Its isolation is described bychromatography and purification (Fiedler, Arzneimittel-Forsch. 4, 213(1953)), and by ion-exchange resins (Erbring et al., U.S. Pat. No.3,238,190). Fractions of escin (also known as aescin) have been purifiedand shown to be biologically active (Yoshikawa M, et al. (Chem PharmBull (Tokyo) 1996 August; 44(8):1454-1464)). Digitonin is anotherdetergent, also being described in the Merck index (12th Ed., entry3204) as a saponin, being derived from the seeds of Digitalis purpureaand purified according to the procedure described by Gisvold et al., J.Am. Pharm. Assoc., 1934, 23, 664; and Rubenstroth-Bauer, Physiol. Chem.,1955, 301, 621.

Other adjuvants or co-adjuvants for use according to certain hereindisclosed embodiments include a block co-polymer or biodegradablepolymer, which refers to a class of polymeric compounds with which thosein the relevant art will be familiar. Examples of a block co-polymer orbiodegradable polymer that may be included in a vaccine composition oran immunological adjuvant include Pluronic® L121 (BASF Corp., MountOlive, N.J.; see, e.g., Yeh et al., 1996 Pharm. Res. 13:1693),

Certain further illustrative embodiments contemplate immunologicaladjuvants that include but are not limited to an oil, which in some suchembodiments may contribute co-adjuvant activity and in other suchembodiments may additionally or alternatively provide a pharmaceuticallyacceptable carrier or excipient. Any number of suitable oils are knownand may be selected for inclusion in vaccine compositions andimmunological adjuvant compositions based on the present disclosure.Examples of such oils, by way of illustration and not limitation,include squalene, squalane, mineral oil, olive oil, cholesterol, and amannide mono-oleate.

Immune response modifiers such as imidazoquinoline are also known in theart and may also be included as adjuvants or coadjuvants in certainpresently disclosed embodiments. As also noted above, one type ofadjuvant or co-adjuvant for use in a vaccine composition according tothe disclosure as described herein may be the aluminium co-adjuvants,which are generally referred to as “alum.” Alum co-adjuvants are basedon the following: aluminium oxy-hydroxide; aluminium hydroxyphosphoate;or various proprietary salts. Alum co-adjuvants are be advantageousbecause they have a good safety record, augment antibody responses,stabilize antigens, and are relatively simple for large-scaleproduction. (Edelman 2002 Mol. Biotechnol. 21:129-148; Edelman, R. 1980Rev. Infect. Dis. 2:370-383.)

Pharmaceutical Compositions

In certain illustrative embodiments, the pharmaceutical composition is avaccine composition that comprises both the recombinant proteinaccording to the disclosure and may further comprise one or morecomponents, as provided herein, that are selected from TLR agonist,co-adjuvant (including, e.g., a cytokine, an imidazoquinoline immuneresponse modifier and/or a dSLIM) and the like and/or a recombinantexpression construct, in combination with a pharmaceutically acceptablecarrier, excipient or diluent.

Illustrative carriers will be nontoxic to recipients at the dosages andconcentrations employed. For vaccines comprising recombinant protein,about 0.01 mu.g/kg to about 100 mg/kg body weight will be administered,typically by the intradermal, subcutaneous, intramuscular or intravenousroute, or by other routes. It will be evident to those skilled in theart that the number and frequency of administration will be dependentupon the response of the host. “Pharmaceutically acceptable carriers”for therapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, sterile salineand phosphate-buffered saline at physiological pH may be used.Preservatives, stabilizers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. For example, sodiumbenzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be addedas preservatives. In addition, antioxidants and suspending agents may beused.

The pharmaceutical compositions may be in any form which allows for thecomposition to be administered to a patient. For example, thecomposition may be in the form of a solid, liquid or gas (aerosol).Typical routes of administration include, without limitation, oral,topical, parenteral (e.g., sublingually or buccally), sublingual,rectal, vaginal, and intranasal (e.g., as a spray). The term parenteralas used herein includes iontophoretic, sonophoretic, passivetransdermal, micro-needle administration and also subcutaneousinjections, intravenous, intramuscular, intrasternal, intracavernous,intrathecal, intrameatal, intraurethral injection or infusiontechniques. In a particular embodiment, a composition as describedherein (including vaccine and pharmaceutical compositions) isadministered intradermally by a technique selected from iontophoresis,micro-cavitation, sonophoresis or micro-needles.

The pharmaceutical composition is formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a patient. Compositions that will be administered toa patient take the form of one or more dosage units, where for example,a tablet may be a single dosage unit, and a container of one or morecompounds of the invention in aerosol form may hold a plurality ofdosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain one ormore of a sweetening agent, preservatives, dye/colorant and flavourenhancer. In a composition intended to be administered by injection, oneor more of a surfactant, preservative, wetting agent, dispersing agent,suspending agent, buffer, stabilizer and isotonic agent may be included.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following carriers or excipients: sterile diluents such as water forinjection, saline solution, preferably physiological saline, Ringer'ssolution, isotonic sodium chloride, fixed oils such as squalene,squalane, mineral oil, a mannide monooleate, cholesterol, and/orsynthetic mono or digylcerides which may serve as the solvent orsuspending medium, polyethylene glycols, glycerin, propylene glycol orother solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. An injectable pharmaceutical composition ispreferably sterile.

In a particular embodiment, a pharmaceutical or vaccine composition ofthe invention comprises a stable aqueous suspension of less than 0.2 umand further comprises at least one component selected from the groupconsisting of phospholipids, fatty acids, surfactants, detergents,saponins, fluorodated lipids, and the like.

It may also be desirable to include other components in a vaccine orpharmaceutical composition, such as delivery vehicles including but notlimited to aluminium salts, water-in-oil emulsions, biodegradable oilvehicles, oil-in-water emulsions, biodegradable microcapsules, andliposomes. Examples of additional immunostimulatory substances(co-adjuvants) for use in such vehicles are also described above and mayinclude N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL-12,GM-CSF, gamma interferon and IL-12.

While any suitable carrier known to those of ordinary skill in the artmaybe employed in the pharmaceutical compositions of this invention, thetype of carrier will vary depending on the mode of administration andwhether a sustained release is desired. For parenteral administration,such as subcutaneous injection, the carrier preferably comprises water,saline, alcohol, a fat, a wax or a buffer. For oral administration, anyof the above carriers or a solid carrier, such as mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,glucose, sucrose, and magnesium carbonate, may be employed.Biodegradable microspheres (e.g., polylactic galactide) may also beemployed as carriers for the pharmaceutical compositions of thisinvention.

Pharmaceutical compositions may also contain diluents such as buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydrates,including glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. Preferably, product may be formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents.

In an illustrative embodiment, the epitope or receptor supporting domainof the recombinant protein, whether derived from a natural or syntheticpolypeptide sequence, should have the capacity to self-assemble intooligomeric multimers under appropriate chemical/environmentalconditions, or to be reduced to monomers under alternative conditions.Ideally, multimerisation domains will assemble into stable multimerswith a discreet number of sub-units, for example dimers, trimers,tetramers, pentamers, etc., such that a product of homogeneous size isgenerated. Without being bound to any particular theory, it is thoughtthat the recombinant synthetic protein, as set forth in Sequence ID: 1,will allow assembly into stable multimers with an insignificant numberof sub-units. Examples of natural polypeptides include, but are notlimited to, leucine zippers, lac repressor protein, streptavidin/avidin,cholera toxin B subunit, Pseudomonas trimerization domain, and viralcapsid proteins.

According to the disclosure, the recombinant proteins, whether growthfactors or parts thereof, cellular receptors or parts thereof, tumorantigens or parts thereof, are related to broad range of either cellularpathways involved in chronic disease or cancers for growth factors andreceptors and to broadest possible range of solid tumors for use oftumor antigens within the said synthetic proteins. The proteins are inthe form of a recombinant protein and may be useful in treating chronicdiseases, for example, breast, lung, bladder, ovarian, vulva, colonic,pulmonary, brain, colorectal, intestinal, head and neck, and esophagealcancers. As different tumor antigens can be expressed and multiplecellular receptors and growth factors over expressed in the saiddiseases, the proteins described hereunder can contain one or moredifferent tumor antigens, one or more different receptors or growthfactors of one or multiple cellular pathways associated with thedisease. These proteins are called “multivalent.”

In the context of the present disclosure, “neutralizing domain” isdefined as a region or regions of either or both member(s) of a specificbinding pair, e.g. a growth factor and its cognate receptor, wherein thebinding of a third molecule that is not a member of the specific bindingpair to the aforementioned region(s) will prevent the subsequent bindingof the two members of the specific binding pair. The third molecule canbe another protein molecule including but not limited to an antibody, orcan be a small non-protein molecule, and can be either natural orsynthetic in origin. The neutralizing domain will normally include thoseregions of the members of the specific binding pair that are in directcontact during binding, and will also include regions out-with saidregions where upon binding of a third molecule introduces sufficientstearic hindrance to prevent the members of the specific binding pairfrom binding directly.

It is well established in the field that specific recognition of aligand by its cognate receptor is defined by an interaction between thebinding site of the receptor and a particular molecular signature(epitope) of the ligand. Thus an antibody that either binds to orotherwise blocks the receptor binding site, or binds to or otherwiseblocks the recognition epitope of the ligand, will prevent ligandreceptor interactions. Such antibodies are described as being“neutralizing.” In the context of the present disclosure it is desirablethat neutralizing antibodies are generated by the host uponadministration of the recombinant protein, and thus the protein sequencemay express or include one or more of all of, or a suitable sequencederived from, a growth factor or tumor antigen such that epitopesrequired for receptor binding are presented in a functional (native)conformation.

In addition to expressing multiple copies of a single tumor antigen,receptor, and/or growth factor, presented as a single tumor antigen,receptor, and/or growth factor or part thereof per physical site, and/oras chains of repetitive tumor antigen, receptor, and/or growth factorsequences (for example, n=1 or more); the protein according to thedisclosure may also include expressions of one or more epitopes orbinding sites from two or more different tumor antigens, receptors,and/or growth factors present as single or as chains at differentpositions within the sequence of the recombinant protein.

In an illustrative embodiment, a protein comprised of a homogeneousrecombinant protein expressing one or more epidermal growth factor (EGF)neutralizing domains is disclosed. The protein is in the form of arecombinant protein and may be useful in treating chronic diseases, forexample, breast, lung, bladder, ovarian, vulva, colonic, pulmonary,brain, colorectal, head and neck, and esophagus cancers. In anillustrative embodiment, the protein is a recombinant protein expressingor including EGF sequences and synthetic polypeptide sequences accordingto the disclosure. In one illustrative embodiment the syntheticpolypeptide sequence is substantially similar to Sequence ID: 1.

In another illustrative embodiment, a protein comprised of a homogeneousrecombinant protein expressing one fibroblast growth factor (FGF) isdisclosed.

In a further illustrative embodiment, the protein is a recombinantprotein expressing or including FGF sequences and synthetic polypeptidesequences according to the disclosure.

In yet a further illustrative embodiment, a protein comprised of ahomogeneous recombinant protein expressing one transforming growthfactor Beta 1 (TGF-β1) is disclosed. In an illustrative embodiment, theprotein is a recombinant protein expressing or including TGF-β1sequences and synthetic polypeptide sequences according to thedisclosure.

In yet another illustrative embodiment, a protein comprised of ahomogeneous recombinant protein expressing one transforming growthfactor-Beta 1 (TGF-β1) is disclosed. In an illustrative embodiment, theprotein is a recombinant protein expressing or including TGF-β1sequences and synthetic polypeptide sequences substantially similar toSequence ID: 2 according to the disclosure.

In another illustrative embodiment, expressing one insulin-like growthfactor-1 (IGF-1) is disclosed. In an illustrative embodiment, theprotein is a recombinant protein expressing or including IGF-1 sequencesand synthetic polypeptide sequences a protein comprised of a homogeneousrecombinant protein expressing one hepatocyte growth factor (HGF) isdisclosed.

In a further illustrative embodiment, the protein is a recombinantprotein expressing or including HGF sequences and synthetic polypeptidesequences according to the disclosure.

In a further illustrative embodiment, a protein comprised of ahomogeneous recombinant protein expressing one Insulin-like growthfactor-1 (IGF-1) and one insulin-like growth factor-2 is disclosed. Inan illustrative embodiment, the protein is a recombinant proteinexpressing or including IGF-1 sequences, IGF-2 sequences and syntheticpolypeptide sequences according to the disclosure.

In yet another illustrative embodiment, a protein comprised of ahomogeneous recombinant protein expressing one vascular endothelialgrowth factor-A (VEGF-A) and one vascular endothelial growth factor-C(VEGF-C) is disclosed. In an illustrative embodiment, the protein is arecombinant protein expressing or including VEGF-A neutralizing domainsequences, VEGF-C sequences and synthetic polypeptide sequencesaccording to the disclosure.

EXAMPLES

Aspects of the disclosure are further described in detail as in thefollowing examples. However, the following examples are not intended tolimit the scope of the disclosure to the precise details of methodologyor construction set forth below. Practical and illustrative embodimentsare illustrated and described in the following examples. However, itwill be appreciated that those skilled in the art may make modificationsand improvements within the spirit and scope of the present disclosure.

Example 1 Construction of a Selectable Carbohydrate-Binding DisplaySystem

A system whereby libraries of mutants could be generated and screenedfor mutants with desirable characteristics was needed that included aphysical linkage between the selectable phenotype and the encodinggenotype of a diverse population of clones. Such a system was developedusing established technologies employed to great effect in the field ofantibody engineering amongst others.

A gene encoding the chosen carbohydrate-binding domain was clonedin-frame and upstream of the minor coat protein gene (gene III) of M13bacteriophage, in both a complete independently functional M13 ‘phage’vector with a suitable selectable marker (M13-K07 ‘phage’), and a‘phagemid’ vector that includes only the F1 packaging region and minorcoat protein-encoding gene from the virus. The former vector whenintroduced into a suitable bacterial host such as E. coli and propagatedappropriately, will generate virus particles that display five copies ofthe chosen carbohydrate-binding domain at one end of the filamentousvirus particle, as N-terminal fusions of the minor coat protein P3(encoded by gene III).

The latter, due to the nature of virus propagation when using aphagemid/helper phage system, and familiar to those practiced in theart, will generate virus particles in which only a small minority of thepopulation will display typically one or less copies of the samecarbohydrate-binding domain-P3 fusion protein.

Clones derived from each vector were propagated under appropriateconditions, well known to those familiar with the technology, and theculture supernatants containing virus particles were screened by ELISAfor binding activity to the major carbohydrate recognised by thecarbohydrate-binding domain. In this example a complex molecule thatincludes a distal galactose group, and which is amenable toimmobilisation by adsorption onto a typical 96 well immunoassay platewas used. In order to bind to the carbohydrate group, it is necessaryfor two or more of the carbohydrate-binding domains to associate in acomplex as a carbohydrate binding pocket is formed between adjacentdomains.

In the former ‘phage’ example, this could potentially be formed betweenadjacent P3 fusion proteins on a single virion, subject to acceptableorientation constraints of the fusion protein, or between separate virusparticles. Using the phagemid system, such associations would mostlikely form between two or more separate virus particles as theproportion of viruses including two or more fusion proteins on a singlevirion is anticipated to be very small.

Upon screening the aforementioned clones, a significantly stronger ELISAsignal was generated from the ‘mono-valent’ phagemid-derived clone thanfrom the penta-valent phage clone, the latter also producing a muchlower titre of infective virus particles. The poor performance of thephage system in this example does not limit the utility of the system inthe process described, but more likely is believed to be a function ofeither steric/orientation constraints imposed on the system by theparticular genetic linkage employed here, and/or infectivityrestrictions imposed by the fusion protein (the P3 minor coat protein isthe mediator of virus infection). It was therefore the phagemid basedsystem that was chosen to generate and select variant mutant proteins.

Example 2 Generation of Mutant Clone Libraries

Libraries of mutant polypeptide-encoding clones were generated from agenetic template derived from a carbohydrate-binding self-assemblingprotein domain. In the present example, the protein domain was selectedfrom the A1B5 group of bacterial holotoxins. Structural and functionalinformation obtained from published data bases such as the Protein DataBase, and the scientific literature, were used to identify regionsand/or specific residues thought potentially or proven to be involved inthe ability of the template protein domain to form stablehomo-pentamers, or to interact with specific carbohydrate-containingmoieties found on the surface of many mammalian cell types. Theseregions/residues were excluded from subsequent mutagenesis and mutantscreening rounds.

Regions or residues defined as above as being potentially amenable tomutation and not involved in desired characteristics were selected andtargeted for rational or random mutagenesis, as deemed appropriate. Inorder to maximise the chances of generating and selectingcarbohydrate-binding variants that were divergent from the templatedomain, mutations were restricted to a limited number of residues inclose proximity for each sequential round of library constructionscreening.

Depending on the region or residue being targeted, mutation was eitherrandom (i.e. including potentially all 20 amino acids), or functionallyrestricted as far as reasonably possible to those residues that displaysimilar bio-physical and/or chemical properties to those found in thetemplate domain.

Characteristics that were considered relevant in such cases included,but were not limited to, side chain size, charge, polarity,hydrophobicity/hydrophilicity, and the ability to participate information of specific secondary structures such as α-helices. Wherepossible/feasible, the residue encoded by the template in any givenposition was omitted from the mutant libraries to avoid selection ofnon-mutants.

Libraries were generated by designing oligonucleotide primers thatincluded both regions of sequence that were homologous to the templategene such that the primer (and specifically its 3′-end) would anneal tothe template DNA under appropriate conditions, and further regions inwhich appropriate degeneracy was included such as to encode a diversityof amino acids in one or more positions.

In the instant example, such oligonucleotides were constructed usingdefined degeneracy familiar to those instructed in the art whereby theDNA base in any diversified position comprised equal quantities of 2-4bases (G, A, T or C) as required. Alternatively, more controlleddiversity is envisaged whereby oligonucleotides generated frompositional mixes of tri-nucleotide phosphoramidites are used such thatthe specific residues (amino acids) and their relative proportions areprecisely controlled at each diversified position.

Where mutations were to be introduced at either terminus, a single PCRreaction was performed (and repeated when necessary) using one or twodegenerate primers in order to introduce the required genetic diversity.The resulting product, which included (primer-derived) flankingrestriction sites, was cloned into the phagemid vector outlined inexample 1 above such that a library of variant proteins was encoded asin-frame genetic fusions to the minor P3 protein-encoding gene (geneIII) of the M13 bacteriophage.

Where is was desired to introduce diversity into region(s) distal to thetermini of the template gene, two pairs of primers were used such thateach pair included one terminal (5′ or 3′) primer that exactly matchedthe template sequence, and one degenerate primer that encoded some, allor none of the required diversity. The 3′-end of the degenerate primerswere designed such that i) each exactly matched the template sequence,and ii) each was exactly complementary to the degenerate primer from thesecond primer pair.

In this way, two PCR products were generated, and then annealed to andlinked to each other via the complementary overlapping sequence derivedfrom the degenerate primers. The resulting product was subsequentlycloned into the phagemid vector as an in-frame genetic fusion with thegene III minor coat protein gene. Successive libraries were either builtupon the outputs from screening and selecting one or more clones fromthe previous library, or were built and screened in parallel from thetemplate or one or more selected mutant clones.

All genetic construct libraries were introduced into host E. coli TG1cells by electroporation using standard methodologies, and transformantsplated onto selective media. Following appropriate incubation, coloniesof bacteria were scraped off plates and stored and/or screened asrequired.

Example 3 Screening of Mutant Libraries

In order to carry out selections of mutants from the libraries, cultures(of libraries) were inoculated into liquid media (2×TY, 100 μg/mlampicillin, 1% glucose) such that sufficient cells to include 100-1000fold representation of the anticipated diversity were included, andsufficient volume to ensure that the OD₆₀₀ was in the region of 0.1.Cultures were then incubated (with shaking) at about 37° C. until theOD₆₀₀ reached 0.4-0.6 (i.e. log phase growth). The cultures were theninfected by the addition of M13 KO7 helper phage at the ratio of ˜20helper phage per bacterial cell (OD₆₀₀ of 1.0 taken to be ˜8.0×10⁸cells/ml). After approximately 30 min static incubation, cells wereincubated for about 30 additional min with shaking. Kanamycin was addedto ˜50 μg/ml and the cultured incubated overnight at ˜30° C. withshaking.

The following morning, cultures were centrifuged for about 25-30 min at8,000 g to pellet cells, and the supernatant removed and retained. Cellpellets were discarded. A 20% volume of 200 mM NaCl, 20% PEG 6,000 wasadded to the culture supernatant, and incubated on ice for about 1 h toprecipitate phage particles. Phage were pelleted at 8,000 g for about25-30 min., and re-suspended in 20 percent of the original volume PBS(phosphate-buffered saline). Again, 20% new volume of 200 mM NaCl, 20%PEG 6,000 was added and the phage incubated on ice for about 25-30 min.The phage were pelleted again at ˜8000 g for 25-30 min, and the pelletre-suspended in ˜2 ml phosphate buffered saline (PBS). The resultingsuspension was transferred to Eppendorf tube(s) and pelleted at maximumspeed for 5 min to pellet any remaining bacterial cells/debris. Thephage suspension was then used for selections.

In order to carry out selections, an immunotube was coated with asuitable antigen such as an immobilizable derivative of galactose, or anatural ligand of the B-subunit, at 1-10 μg/ml (typically 5 ml)overnight at 4-8° C. or at room temperature for 1 h. After washing 3-5times with PBS (by simply pouring into the immunotube and pouring outagain), the tube was blocked by the addition of MPBS (PBS containing 2%milk powder) for 1-2 h at 37° C. The MPBS was washed out as describedabove, and approximately 1×10¹¹ to 1×10¹² or more phage particles added.

The volume was made up to ˜5 ml, and the immunotube sealed e.g. withpara-film. It was then ‘tumbled’ (end-over-end) for about 30 min, thenincubated standing for about 90 min to allow those phage that display aprotein from the mutant library that is able to recognise theimmobilised ligand, to bind to it. Phage that had not bound, or wereonly weakly bound, were removed by washing. The stringency of theselection was varied as required by the number of washes used, the useof PBST (PBS containing 0.1% Tween 20) to wash, or adjusting the coatingconcentration of ligand.

Bound phage particles were eluted from the immunotube by adding 1 ml 100mM triethylamine (TEA) and tumbling for a maximum of 10 min (longerincubation adversely affects phage viability), then pouring immediatelyinto 0.5 ml 1 M Tris-HCl (pH 7.4) to neutralise. About 0.75 ml elutedphage was added to ˜10 ml log phase culture of a suitable E. coli strainsuch as TG1 (Agilent). The culture was incubated at about 37° C. withoutshaking to allow infection. Serial dilutions of a sample of the infectedcells were spread onto small TYE plates containing 100 μg/ml ampicillinand 1% glucose. The remaining cells were plated onto larger bioassayplates with the same media. All were incubated overnight at about 30° C.Of the other 0.75 ml phage, ˜75 μl were infected as above into ˜1 ml logphase E. coli HB2151 cells and serial dilutions plated out as andincubated as above. The remaining phage were stored at about −80° C. asa glycerol stock (˜15% glycerol).

The following day large bio-assay plates were scraped to remove thecells, which were then stored as glycerol stocks or grown and ‘rescued’with M13 KO7 helper phage as described earlier, to prepare an enrichedpopulation of phage for the next round of selection. Typically two tothree sequential rounds of selection were carried out. Upon completionof selections, individual colonies from the small serial dilution platesof HB2151 cells were picked into 96 well culture plates containing 100μl/well 2×TY with 100 μg/ml ampicillin, and incubated with shakingovernight at 37° C. The following day, approximately 5 μl culture fromeach well was inoculated into a fresh plate with ˜150 μl media/well, andincubated at about 37° C. with shaking for about 2 hours. Additionalmedia containing 1 M IPTG was added to give a final concentration of 1mM IPTG, and the plate incubated with shaking overnight at 30° C. InHB2151 cells, induction with IPTG results in the expression of solublemutant proteins including a detectable c-myc peptide tag, rather thanproteins fused to a component of a virus particle as is the case with E.coli TG1 cells. The initial 96 well culture plate was stored at about−80° C.

The next day, culture supernatants from the induced 96 well plate wereassayed for binding to immobilised carbohydrate using typical ELISAprotocols, and binding detected with a readily available HRP-labelledanti-c-Myc antibody (FIG. 1). Clones that were strongly positive forbinding to GM1 are shown in shaded cells. Positive and negative controlsare boxed.

Culture supernatants from clones that were strongly positive by bindingELISA were further analysed by SDS PAGE and Western blot to assess thepresence of pentamers and other multimeric states as illustrated in FIG.2. Samples were not reduced or boiled prior to electrophoresis, andprotein was detected with HRP-labelled anti-c-Myc antibody. In theexample shown, clones C1 and C3 are positive controls that readilyassemble into pentamers of ˜60 kDa. Clone C2 is a negative control thatruns as a 12 kDa monomer under the conditions used. Clones A1 and A3 canbe seen to run primarily as pentamers, whereas a significant proportionof clone B12 forms monomers. The remaining clones form either onlymonomer (B1, B7) or form many multimeric forms.

Example 4 Compilation of Mutations

In order to assess the capacity of the maximum number of residues toaccommodate mutations, several different libraries each targetingdifferent residues or regions of the template, were constructed andscreened. As a result, a large number of clones were identified thatexhibited the desired selection criteria of binding to immobilisedcarbohydrate derivatives and of forming predominantly pentamers, howevereach of which included a number of unique mutations/differences asillustrated in FIG. 3A. In order to generate clones with potentiallyenhanced characteristics it was necessary to combine these variedsequences into a smaller number of clones.

As the mutant generation and selection process had identified severalpositions where three or more different residues could be accommodated,a total of four combination mutants (A-D) were designed as illustratedin FIG. 3B. The four clones containing combined mutations wereexpressed, and the synthetic proteins evaluated for their bindingcompared to appropriate controls by ELISA as illustrated in FIG. 4. Itwas found that two of the clones (A and C) had only weak binding,however the other two (B and D) were able to bind to GM1 very strongly.This indicates that mutations derived from separate clones can becombined successfully, however the enhancement or even retention or ofselected binding characteristics is not guaranteed.

Example 5 Stabilization of Homo-Pentamers

In their native fully functional form, the bacterial holotoxinsdescribed by the group name ‘A1B5’ comprise five B-subunits and a singleA-subunit. The B subunits assemble into a pentameric ring with a central‘pore’. A single A-subunit sits atop this ring and projects down intothe void as illustrated in FIG. 5. The A-subunit itself comprises 2distinct domains. The A1 domain, which is released when the holotoxin istranslocated into a cell, contains the enzymatic activity associatedwith the toxicity of the protein complex. It is held as a part of theholotoxin by the A2 domain. This has an alpha-helical structure in whichthe A1 domain sits at the N-terminus, and the C-terminus projects intothe pore formed by the 5 B-subunits. Some residues of the C-terminus ofA2 form interactions with residues of the B-subunits. As there areconfirmed interactions between specific residues of the B-subunits andthe A2 domain (Zhang R. G., et al (1995)), it is reasonable to suggestthat the presence of the A-subunit, and specifically the A2 domain,contributes to stabilizing the pentameric B-subunit ring. Due to thestructural and functional distinctions between the two A-subunitdomains, without being bound to any particular theory, it is thoughtthat the A2 domain alone would confer this stabilizing effect on aB-subunit pentamer. Similarly, as it is known that B-subunits are ableto form pentameric rings in the complete absence of the A-subunit, it isprobable that the A2 domain could associate with, and therefore exert astabilizing effect on B-subunit pentamers by either being co-expressedwith the B-subunits, or by being added exogenously.

It is therefore proposed that a synthetic or non-naturally occurringpolypeptide, that shares a sufficiently similar conformation toholotoxin B-subunits, that is able to assemble into multimers includingpentameric rings, and in which the residues corresponding to those thatinteract with A2 in holotoxin B-subunits are the same or share the samebio-physical properties, then such a synthetic protein pentamer wouldexperience a similar stabilizing benefit. Moreover, it is suggested thatin the absence of a natural A2 domain, a synthetic polypeptide could beisolated from a suitable polypeptide library using technologies familiarto those practiced in the art such as ‘phage display’, that would bindat least in part to the exposed surfaces of two or more natural orsynthetic monomers when assembled into a pentamer, and so stabilize thestructure.

As naturally occurring A2 domains support a structurally independent A1domain that does not in itself interact with any other component of theholotoxin, then it is probable that an A2 domain or a functionalsynthetic equivalent could provide a means of anchoring anotherpolypeptide domain. Without being bound to any particular theory, it isthought that non-naturally occurring polypeptide, that shares asufficiently similar conformation to polypeptide described in SequenceID: 1 contributes by itself to form pentameric rings exhibiting astabilized pentameric ring structure allowing for the attachment ofepitopes as described within this disclosure.

Example 6

Reference is to FIG. 7 for subsequent mutation events. A major binderfrom the screening that targeted mutations (library 6) in a confinedregion (the β3 strand) of CTB was selected. Starting off with thistemplate (L6e5) one mutagenesis intermezzo without library constructionand screening was carried out. It involved the same DNA methodology asforth in EXAMPLE 2, i.e. generation of PCR products and subsequentannealing/linking via complementary overlapping sequence. The β5-α2 loop(positions 55-64), a ‘permissive’ site proposed by Backstrom et al.(Gene, 1994), was addressed for inserting peptides. Some non-humanepitope peptides were tested and substitution with FVSTLQAA passedcriteria (GM1 binding and pentamer formation). The DNA sequence of thiscarrier was the template for generating the next library, targeting theflexible ends of the protein (positions 1-4 and 102-3). Screening diddeliver mutants meeting criteria and the DNA sequence of one of them wasused as template in a library targeting two untouched middle regions(approximate β1-β2 and α2-β5 loops). Once more did screening releaseGM1-binding pentameric proteins. One of them is notable (5B5, 20%changed υ CTB), (FIGS. 8A and 8B, SEQUENCE-3).

Synthetic carrier 5B5 was assessed in ELISAs with anti-CTB antibodies.Detection by a monoclonal was significantly impaired (FIG. 9A).Polyclonal antibodies (three suppliers tested) exhibited a trend towardsreduced detection of 5B5. This is illustrated for one of them (a sheepserum source) with normalised amounts of carrier (FIG. 9B).

In addition, we propose the practicality of the glycine (G) to aspartate(D) mutation at position 33 of the carrier/subunit sequence (Jobling &Holmes, Mol. Microbiol. 1991). The G33D mutation disables gangliosideGM1 binding but subunits still form a pentamer. This was confirmed forbinders selected for next mutation round (FIG. 7), but its G33D DNAversion was obviously not used as next-round template, and the final 5B5(FIGS. 7 and 8: the introduced negatively charged aspartate gives it aslower electrophoretic mobility). GM1 is present on all cells. Asynthetic carrier that is not able to bind GM1 will beavailable/presented to immune cells in a higher dosage. Moreover,GM1-binding inability, 20% sequence difference, impaired detection withanti-CTB antibodies, and (pending, see below) absence of assembly withCTA makes it distinct from CTB.

During processing of the last two libraries, precursor carrier d8_G33D(12% substituted) was fused to a growth factor, this time hFGF2, andHis6-tag purified recombinant conjugate was used to immunise mice, viai.m. administration. Significant IgG antibody responses against FGF2were obtained (FIG. 10). Immunisations of 5B5 and follow-ups, with andwithout G33D, fused to hFGF2 (SEQUENCE-4) are planned (pendingpurification and characterisation).

Example 7

The cholera holotoxin is an AB5 hexamer, i.e. CTA non-covalentlyinteracts with the CTB pentamer, entering cells via ganglioside GM1 cellsurface receptor. The proposed synthetic carrier pentamer is distinctform CTB, besides at least 20% sequence difference and its inability tobind GM1, if it does not bind (the CTA2 component) of CTA. Tinker et al.(Infect. Immun. 2003) have shown that substituting a single residue inCTB (I74D or T78D, in the region interacting with CTA2) disablesholotoxin assembly. Synthetic carrier 5B5 with these single mutationsstill formed pentamers, as shown in FIGS. 11A and 11B for I74D, butabsence of interaction with CTA2 in vitro has not been established yet.

Example 8

It is anticipated that a monomeric carrier will be useful to fuse to andpurify complicated antigens, like dimeric TGFβ1 growth factor. With thelatter it will still be exposed to the immune cells/system as arelatively large (four entities) complex. Modelling of CTB has revealedexposed hydrophobic residues potentially involved in subunitinteraction/multimerisation. By changing them to hydrophilic/polar aminoacids (e.g. F25R, L31E, A32Q, L77N) the current pentameric syntheticcarrier (5B5), may well be converted to a monomeric version.

Although the devices, systems, and methods have been described andillustrated in connection with certain embodiments, many variations andmodifications will be evident to those skilled in the art and may bemade without departing from the spirit and scope of the disclosure. Thediscourse is thus not to be limited to the precise details ofmethodology or construction set forth above as such variations andmodification are intended to be included within the scope of thedisclosure.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

What is claimed is:
 1. A recombinant synthetic protein comprising: amonomeric sequence that is able to assemble into stable homomultimers.2. A recombinant protein according to claim 1, wherein each monomericsequence includes one or more additional polypeptide sequences.
 3. Arecombinant protein according to claim 2, wherein said polypeptidesequences are selected from the group comprising: growth factors,receptors, parts thereof, and combinations thereof.
 4. The recombinantprotein according to claim 2, wherein said additional polypeptidesequences includes at least one immunogenic sequence.
 5. The recombinantprotein according to claim 3, wherein said growth factor includes atransforming growth factor (TGF).
 6. The recombinant protein accordingto claim 3, wherein said at least said portion of said growth factorincludes a neutralizing domain of a growth factor.
 7. The recombinantprotein according to claim 3, wherein said at least said portion of saidsignalling includes a full length or part thereof of one or more of thefollowing growth factors selected from the group consisting of IGF-1,IGF-2, FGF1, FGF2, TGF-α, TGF-β, VEGF-A, VEGF-B, VEGF-C, VEGF, D, PDGF,NGF, EGF, HGF, BMP's, PDL1 and IL's 1-6.
 8. The recombinant protein.according to claim 3, wherein said additional polypeptide sequencesincludes a neutralizing domain of at least two different growth factorspresent in said synthetic protein.
 9. The recombinant protein accordingclaim 3, wherein at least a portion of said growth factor includes afull length or neutralizing domain of one or more growth factors in saidprotein as single domain or as two or more multiple repeats.
 10. Therecombinant protein according claim 9, wherein at least a portion ofsaid growth factor or a neutralizing, domain thereof is separated fromthe recombinant protein by a spacer.
 11. A recombinant protein accordingclaim 10, wherein said spacer comprises in part a growth factor orneutralizing domain thereof.
 12. A recombinant protein according claim10, wherein said spacer includes one or more host T-cell epitopes.
 13. Aprocess of preparing a multivalent molecule comprising: assemblingmultimers from monomeric sub-units to form a synthetic protein includingone or more growth factors or parts thereof.
 14. A process of preparinga vaccine formulation comprising: mixing one or more single monovalentmultimers together preparing a multivalent vaccine including a syntheticprotein including one or more growth factors or parts thereof.
 15. Aprocess for treating a patient comprising: administering an immunogenicdose to said patient of one or more synthetic proteins according toclaim 3 during a treatment period.
 16. A recombinant protein,comprising: a polypeptide sequence; and a first sequence expressing atleast a portion of a tumor antigen within said polypeptide sequence. 17.The recombinant protein according to claim 16, wherein said polypeptidesequence includes an immunogenic sequence.
 18. The recombinant proteinaccording to claim 16, wherein said polypeptide sequence includes animmunogenic synthetic polypeptide sequence.
 19. The recombinant proteinaccording to claim 16, wherein said at least said portion of said tumorantigen includes a full length or a portion of one or more differenttumor antigens present in said synthetic protein.
 20. The recombinantprotein according claim 16, wherein at least said portion of said tumorantigen includes a full length or a portion of one or more tumorantigens in said synthetic protein as single epitopes or in multiplerepeats.
 21. The recombinant protein according to claim 16, furthercomprising at least one additional sequence expressing at least aportion of a growth factor.
 22. The recombinant protein according toclaim 21, wherein said portion of said growth factor includes a fulllength or part thereof of one or more of the following growth factorsselected from the group consisting of IGF1,IGF-2, FGF1, FGF2, TGF-α,TGF-β, VEGF-A, VEGF-B, VEGF-C, VEGF, D, PDGF, NGF, EGF, HGF, BMP's, andIL's 1-6.
 23. The recombinant protein according to claim 22, whereinsaid portion of said growth factor includes a full length orneutralizing portion of at least two different growth factors present insaid synthetic protein.
 24. The recombinant protein according claim 22,wherein said at least said portion of said growth factor includes a fulllength or neutralizing portion of one or more growth factors in saidsynthetic protein as single epitopes or as two or more tandem repeats.25. The recombinant protein according claim 22, wherein said at leastsaid portion of said growth factor or a neutralizing portion thereof isseparated from the remaining synthetic protein by a peptide spacer. 26.The recombinant protein according to claim 16, further comprising apolypeptide sequence that is substantially similar to Sequence ID: 1.27. The recombinant protein according to claim 26, expressing at least aportion of a receptor along said polypeptide sequence, wherein said atleast said portion of said receptor includes a full length or a portionof two to four different receptors present in said protein.
 28. Therecombinant protein according to claim 26, further comprising a secondsequence expressing at least a portion of a growth factor along saidpolypeptide sequence, wherein said at least said portion of said growthfactor includes a full length or part thereof of one or more of thefollowing growth factors selected from the group consisting of IGF-1,IGF-2, FGF1, FGF2, TGF-β, VEGF-A, VEGF-B, VEGF-C, VEGF, D, PDGF, NGF,EGF, HGF, BMP's, and IL's 1-6.
 29. The recombinant protein according toclaim 28, wherein said at least said portion of said growth factorincludes a full length or neutralizing portion of one or more growthfactors in said synthetic protein as single epitopes or as two or moretandem repeats.